Method and apparatus for analysis and sorting of polynucleotides based on size

ABSTRACT

The invention relates to a microfabricated device and methods of using the device for analyzing and sorting of single polynucleotides, e.g. by size, according to an optical signal measured within a detection region of the device. An optical signal such as fluorescence from a reporter molecule associated with the polynucleotide molecules can be used to determine polynucleotide size or to direct selected polynucleotides into one or more selected branch channels of the device.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/026,693, filed Sep. 25, 1996, incorporated herein by reference inits entirety.

The U.S. Government may have certain rights in this invention pursuantto Grant No. DAAH04-96-1-0141 awarded by the Army.

FIELD OF THE INVENTION

The present invention relates in general to a method of analyzing andsorting polynucleotides (e.g., DNA) by size. In particular, theinvention relates to a method of analyzing and/or sorting individualpolynucleotide molecules in a microfabricated device by measuring thesignal of an optically-detectable (e.g., fluorescent) reporterassociated with the molecules.

REFERENCES

Aine, H. E., et al., U.S. Pat. No. 4,585,209 (1986).

Baker, D. R., in CAPILLARY ELECTROPHORESIS, John Wiley & Sons, New York,1995.

Ballantyne, J. P., et al., J. Vac. Sci. Technol. 10:1094 (1973).

Castro, A., et al., Anal. Chem. 85:849-852 (1993).

Goodwin, P. M., et al., Nucleic Acids Research 21-(4) :803-806 (1993)

Gravesen, P., et al., U.S. Pat. No. 5,452,878 (1995).

Haugland, R. P., in HANDBOOK OF FLUORESCENT PROBES AND RESEARCHCHEMICALS, 5th Ed., Molecular Probes, Inc., Eugene, Oreg. (1992).

Keller, R. A., et al., GB Patent No. 2,264,496 (Oct. 10, 1995).

Krutenat, R. C., in KIRK-OTHMER ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY, 3rdEd., John Wiley & Sons, New York, Vol. 15, 241-274.

O'Connor, J. M., U.S. Pat. No. 4,581,624 (1986) van Lintel, H. T. G.,U.S. Pat. No. 5,271,724 (1993).

Wise, K. D., et al., U.S. Pat. No. 5,417,235 (1995).

BACKGROUND OF THE INVENTION

Identification and separation of nucleic acid fragments by size, such asin sequencing of DNA or RNA, is a widely used technique in many fields,including molecular biology, biotechnology, and medical diagnostics. Themost frequently used method for such separation is gel electrophoresis,in which different sized charged molecules are separated by theirdifferent rates of movement through a stationary gel under the influenceof an electric current. Gel electrophoresis presents severaldisadvantages, however. The process can be time consuming, andresolution is typically about 10%. Efficiency and resolution decrease asthe size of fragments increases; molecules larger than 40,000 aredifficult to process, and those larger than 10 million base pairs cannotbe distinguished.

Methods have been proposed for determination of the size of nucleic acidmolecules based on the level of fluorescence emitted from moleculestreated with a fluorescent dye (Keller, et al., 1995; Goodwin, et al.,1993; Castro, et al., 1993). Castro describes the detection ofindividual molecules in samples containing either uniformly sized (48Kbp) DNA molecules or a predetermined 1:1 ratio of molecules of twodifferent sizes (48 Kbp and 24 Kbp). A resolution of approximately12-15% was achieved between these two sizes. There is no discussion ofsorting or isolating the differently sized molecules.

In order to provide a small diameter sample stream, Castro uses a“sheath flow” technique wherein a sheath fluid hydrodynamically focusesthe sample stream from 100 μm to 20 μm. This method requires that theradiation exciting the dye molecules, and the emitted fluorescence, musttraverse the sheath fluid, leading to poor light collection efficiencyand resolution problems caused by lack of uniformity. Specifically, thismethod results in a relatively poor signal-to-noise ratio of thecollected fluorescence, leading to inaccuracies in the sizing of the DNAmolecules.

Goodwin mentions the sorting of fluorescently stained DNA molecules byflow cytometry. This method, however, employs costly and cumbersomeequipment, and requires atomization of the nucleic acid solution intodroplets, with the requirement that each droplet contains at most oneanalyte molecule. Furthermore, the flow velocities required forsuccessful sorting of DNA fragments were determined to be considerablyslower than used in conventional flow cytometry, so the method wouldrequire adaptations to conventional equipment. Sorting a usable amount(e.g., 100 ng) of DNA using such equipment would take weeks, if notmonths, for a single run, and would generate inordinately large volumesof DNA solution requiring additional concentration and/or precipitationsteps.

It is thus desirable to provide a method of rapidly analyzing andsorting differently sized nucleic acid molecules with high resolution,using simple and inexpensive equipment. A short optical path length isdesirable to reduce distortion and improve signal-to-noise of detectedradiation. Ideally, sorting of fragments can be carried out using anysize-based criteria.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes a microfabricated devicefor sorting reporter-labelled polynucleotides or polynucleotidemolecules by size. The device includes a chip having a substrate intowhich is microfabricated at least one analysis unit. Each analysis unitincludes a main channel, having at one end a sample inlet, having alongits length a detection region, and having, adjacent and downstream ofthe detection region, a branch point discrimination region. The analysisunit further includes a plurality of branch channels originating at thediscrimination region and in communication with the main channel, ameans for passing a continuous stream of solution containing themolecules through said detection region, such that on average only onemolecule occupies the detection region at any given time, a means formeasuring the level of reporter from each molecule within the detectionregion, and a means for directing the molecule to a selected branchchannel based on the level of reporter.

In one general embodiment, the directing or sorting means includes apair of electrodes effective to apply an electric field across thediscrimination region, where the applied field is effective to direct aparticular molecule into a selected branch channel based on the amountof reporter signal detected from that molecule.

In another general embodiment, a flow of molecules is maintained throughthe device via a pump or pressure differential, and the directing meanscomprises a valve structure at the branch point effective to permit themolecule to enter only one of the branch channels.

In still another general embodiment, a flow of molecules is maintainedthrough the device via a pump or pressure differential, and thedirecting means comprises, for each branch channel, a valve structuredownstream of the branch point effective to allow or curtail flowthrough the channel.

In a related general embodiment, a flow of molecules is maintainedthrough the device via a pump or pressure differential, and thedirecting means comprises, for each branch channel, a pressure adjustingmeans at the outlet of each branch channel effective to allow or curtailflow through the channel.

A device which contains a plurality of analysis units may furtherinclude a plurality of manifolds, the number of such manifolds typicallybeing equal to the number of branch channels in one analysis unit, tofacilitate collection of molecules from corresponding branch channels ofthe different analysis units.

In preferred embodiments, the device includes a transparent (e.g.,glass) cover slip bonded to the substrate and covering the channels toform the roof of the channels. The channels in the device are preferablybetween about 1 μm and about 10 μm in width and between about 1 μm andabout 10 μm in depth, and the detection region has a volume of betweenabout 1 fl and about 1 pl.

The exciting means may be, for example, an external laser, a diode orintegrated semiconductor laser or a high-intensity lamp (e.g., mercurylamp).

The measuring means may be, for example, a fluorescence microscope inconnection with an intensified (e.g., SIT) camera, an integratedphotodiode, or the like.

In another aspect, the invention includes a method of isolatingpolynucleotides having a selected size. The method includes A) flowing acontinuous stream of solution containing reporter-labeledpolynucleotides through a channel comprising a detection region having aselected volume, where the concentration of the molecules in thesolution is such that the molecules pass through the detection regionone by one, B) determining the size of each molecule as it passesthrough the detection region by measuring the level of the reporter, C)in the continuous stream of solution, diverting (i) molecules having theselected size into a first branch channel, and (ii) molecules not havingthe selected size into a second branch channel, and D) collectingpolynucleotides diverted into the first branch channel.

In general preferred embodiments, the concentration of polynucleotidesin the solution is between about 10 fM and about 1 nM and the detectionregion volume is between about 1 fl and about 1 pl.

The determining typically includes quantitating an optical signal, suchas a fluorescence signal, from an optical reporter, such as afluorescent moiety, associated with the polynucleotides. Exemplaryfluorescent moieties are fluorescent reporters selected from the groupconsisting of POPO, BOBO, YOYO, and TOTO.

In a general embodiment, the diverting includes the transientapplication of an electric field effective to bias (i) a molecule havingthe selected size (e.g., between about 100 bp and about 10 mb) to enterthe first branch channel, and (ii) a molecule not having the selectedsize to enter the second branch channel.

The method may be applied to diverting a molecule having the selectedsize into the first branch channel, wherein the diverting includesblocking the flow in the second branch channel such that the continuousstream of solution carries the molecule having the selected size intothe first branch channel. Alternatively or in addition, the method maybe applied for diverting a molecule not having the selected size intothe second branch channel, wherein the diverting includes blocking theflow in the first branch channel such that the continuous stream ofsolution carries the fragment not having the selected size into thesecond branch channel.

The diverting may include a mechanical switch effective to direct (i) afragment having the selected size to enter the first branch channel, and(ii) a fragment not having the selected size to enter the second branchchannel.

In yet another aspect, the invention includes a method of sizingpolynucleotides in solution. This method includes: A) flowing acontinuous stream of solution containing reporter-labeledpolynucleotides through a microfabricated channel comprising a detectionregion having a selected volume, where the concentration of themolecules in the solution is such that most molecules pass through thedetection region one by one, and B) determining the size of eachmolecule as it passes through the detection region by measuring thelevel of the reporter.

In still another aspect, the invention includes a microfabricated devicefor sorting reporter-labelled cells by the level of reporter theycontain. The device includes a chip having a substrate into which ismicrofabricated at least one analysis unit. Each analysis unit includesa main channel, having at one end a sample inlet, having along itslength a detection region, and having, adjacent and downstream of thedetection region, a branch point discrimination region. The analysisunit further includes a plurality of branch channels originating at thediscrimination region and in communication with the main channel, ameans for passing a continuous stream of solution containing the cellsthrough said detection region, such that on average only one celloccupies the detection region at any given time, a means for measuringthe level of reporter from each cell within the detection region, and ameans for directing the cell to a selected branch channel based on thelevel of reporter.

In one embodiment, a flow of cells is maintained through the device viaa pump or pressure differential, and the directing means comprises avalve structure at the branch point effective to permit each cell toenter only one of the branch channels.

In another general embodiment, a flow of cells is maintained through thedevice via a pump or pressure differential, and the directing meanscomprises, for each branch channel, a valve structure downstream of thebranch point effective to allow or curtail flow through the channel.

In a related general embodiment, a flow of cells is maintained throughthe device via a pump or pressure differential, and the directing meanscomprises, for each branch channel, a pressure adjusting means at theoutlet of each branch channel effective to allow or curtail flow throughthe channel.

A device which contains a plurality of analysis units may furtherinclude a plurality of manifolds, the number of such manifolds typicallybeing equal to the number of branch channels in one analysis unit, tofacilitate collection of cells from corresponding branch channels of thedifferent analysis units.

In preferred embodiments, the device includes a transparent (e.g.,glass) cover slip bonded to the substrate and covering the channels toform the roof of the channels. The channels in the device are preferablybetween about 20 μm and 500 μm in width and between about 20 μm and 500μm in depth, and the detection region has a volume of between about 10pl and 100 nl.

The exciting means may be, for example, an external laser, a diode orintegrated semiconductor laser or a high-intensity lamp (e.g., mercurylamp).

The measuring means may be, for example, a fluorescence microscope inconnection with an intensified (e.g., SIT) camera, an integratedphotodiode, or the like.

In another aspect, the invention includes a method of isolating cellshaving a selected amount of bound optically-detectable (e.g.,fluorescent) reporter. The method includes A) flowing a continuousstream of solution containing reporter-labeled cells through a channelcomprising a detection region having a selected volume, where theconcentration of the cells in the solution is such that the moleculespass through the detection region one by one, B) determining the amountof reporter on each cell as it passes through the detection region, C)in the continuous stream of solution, diverting (i) cells having theselected amount of reporter into a first branch channel, and (ii) cellsnot having the selected amount of reporter into a second branch channel,and D) collecting cells diverted into the branch channels.

The method may be applied to diverting a cell having the selected amountof reporter into the first branch channel, wherein the divertingincludes blocking the flow in the second branch channel such that thecontinuous stream of solution carries the cell having the selectedamount of reporter into the first branch channel. Alternatively or inaddition, the method may be applied for diverting a cell not having theselected amount of reporter into the second branch channel, wherein thediverting includes blocking the flow in the first branch channel suchthat the continuous stream of solution carries the fragment not havingthe selected amount of reporter into the second branch channel.

The diverting may include a mechanical switch effective to direct (i) acell having the selected amount of reporter to enter the first branchchannel, and (ii) a cell not having the selected amount of reporter toenter the second branch channel.

The method may be applied to any procaryotic or eukaryotic cells, suchas bacterial cells, mammalian cells, and the like. The method isparticularly useful for the sorting of mammalian (e.g., human) bloodcells, such as peripheral blood mononuclear cells (PBMCs), based on thepatterns of expression of various antigens, such as HLA DR, CD3, CD4,CD8, CD11a, CD11c, CD14, CD16, CD20, CD45, CD45RA, CD62L, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleic acid sorting device in accordance with oneembodiment of the invention;

FIG. 2 shows a partial perspective view of a nucleic acid sortingdevice, showing a sample solution reservoir and sample inlet;

FIG. 3A shows one embodiment of a detection region used in a nucleicacid sorting device, having an integrated photodiode detector;

FIG. 3B shows another embodiment of a detection region, having anintegrated photodiode detector, and providing a larger detection volume(than the embodiment of FIG. 3A);

FIG. 4A shows one embodiment of a discrimination region used in anucleic acid sorting device, having electrodes disposed within thechannels for electrophoretic discrimination;

FIG. 4B shows another embodiment of a discrimination region used in anucleic acid sorting device, having electrodes disposed forelectroosmotic discrimination;

FIGS. 4C and 4D show two further embodiments of a discrimination region,having valves disposed for pressure electrophoretic separation, wherethe valves are within the branch point, as shown in 4C, or within thebranch channels, as shown in 4D;

FIGS. 5A-5D show initial steps in photolithographic microfabrication ofa nucleic acid sorting device from a silicon wafer, usingphotolithography and several stages of etching;

FIGS. 6A-6B show one embodiment of a valve within a branch channel of anucleic acid sorting device, and steps in fabrication of the valve; and

FIG. 7 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofpolynucleotide or cell sorting.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below have the following meanings unless indicated otherwise.

The term “polynucleotide” as used herein refers to a polymeric moleculehaving a backbone that supports bases capable of hydrogen bonding totypical polynucleotides, where the polymer backbone presents the basesin a manner to permit such hydrogen bonding in a sequence specificfashion between the polymeric molecule and a typical polynucleotide(e.g., single-stranded DNA). Such bases are typically inosine,adenosine, guanosine, cytosine, uracil and thymidine. Polymericmolecules include double and single stranded RNA and DNA, and backbonemodifications thereof, for example, methylphosphonate linkages.

II. Overview of Invention

According to one aspect of the present invention, polynucleotides, e.g.,DNA, can be sorted dynamically in a continuous flow stream ofmicroscopic dimensions based on the molecules' molecular weight using amicrofabricated polynucleotide sorting device. The polynucleotides,suspended in a suitable carrier fluid (e.g., ddH2O or TE), areintroduced into an inlet end of a narrow channel in the sorting device.The molecular weight of each molecule is calculated from the intensityof signal from an optically-detectable reporter incorporated into orassociated with the polynucleotide molecule as the molecule passesthrough a “detection window” or “detection region” in the device.Molecules having a molecular weight falling within a selected range arediverted into a selected output or “branch” channel of the device. Thesorted polynucleotide molecules may be collected from the outputchannels and used in subsequent manipulations.

According to another aspect of the invention, a device such as describedabove, but not necessarily including components for sorting themolecules, can be used to simply quantitate the size range ofpolynucleotides in a sample, and store or feed this information into aprocessor or computer for subsequent analysis or display, e.g., as asize distribution histogram. Such a device enables the generation of thetype of polynucleotide fragment length data now commonly obtained fromanalytical gels, such as agarose or polyacrylamide gels, in a fractionof the time required for preparation and analysis of gels, and using asubstantially smaller amount of sample.

According to yet another aspect of the invention, a sorting device suchas described above can be used to sort cells based on the level ofexpression of selected cell markers, such as cell surface markers, in amanner similar to that currently employed using fluorescence-activatedcell sorting (FACS) machines.

III. Microfabricated Polynucleotide Sorting Device

FIG. 1 shows an embodiment of a microfabricated polynucleotide sortingdevice 20 in accordance with the invention. The device is preferablyfabricated from a silicon microchip 22. The dimensions of the chip arethose of typical microchips, ranging between about 0.5 cm to about 5 cmper side and about 0.1 mm to about 1 cm in thickness. The devicecontains a solution inlet 24, two or more solution outlets, such asoutlets 26 and 28, and at least one analysis unit, such as the unit at30.

Each analysis unit includes a main channel 32 having at one end a sampleinlet 34, and downstream of the sample inlet, a detection region 36, anddownstream of the detection region 36 a discrimination region 38. Aplurality of branch channels, such as channels 40 and 42, are in fluidcommunication with and branch out from the discrimination region. Thedimensions of the main and branch channels are typically between about 1μm and 10 μm per side, but may vary at various points to facilitateanalysis, sorting and/or collection of molecules.

In embodiments such as shown in FIG. 1, where the device contains aplurality of analysis units, the device may further contain collectionmanifolds, such as manifolds 44 and 46, to facilitate collection ofsample from corresponding branch channels of different analysis unitsfor routing to the appropriate solution outlet. The manifolds arepreferably microfabricated into different levels of the device, asindicated by the dotted line representing manifold 46. Similarly, suchembodiments may include a sample solution reservoir, such as reservoir48, to facilitate introduction of sample into the sample inlet of eachanalysis unit.

Also included with the device is a processor, such as processor 50. Theprocessor can be integrated into the same chip as contains the analysisunit(s), or it can be separate, e.g., an independent microchip connectedto the analysis unit-containing chip via electronic leads, such as leads52 (connected to the detection region(s) and 54 (connected to thediscrimination region(s)).

As mentioned above, the device may be microfabricated with a samplesolution reservoir to facilitate introduction of a polynucleotidesolution into the device and into the sample inlet of each analysisunit. With reference to FIG. 2, the reservoir is microfabricated intothe silicon substrate of the chip 62, and is covered, along with thechannels (such as main channel 64) of the analysis units, with a glasscoverslip 66. The device solution inlet comprises an opening 68 in thefloor of the microchip. The inlet may further contain a connector 70adapted to receive a suitable piece of tubing, such as liquidchromatography or HPLC tubing, through which the sample may be supplied.Such an arrangement facilitates introducing the sample solution underpositive pressure, to achieve a desired flow rate through the channelsas described below.

Downstream of the sample inlet of the main channel of each analysis unitis the detection region, designed to detect the level of anoptically-detectable reporter associated with polynucleotides present inthe region. Exemplary embodiments of detection regions in devices of theinvention are shown in FIGS. 3A and 3B.

With reference to FIG. 3A, each detection region is formed of a portionof the main channel of an analysis unit and a photodiode, such asphotodiode 72, located in the floor of the main channel. In thisembodiment, the area detectable by the detection region is the circularportion each channel defined by the receptive field of the photodiode inthat channel. The volume of the detection region is the volume of acylinder with a diameter equal to the receptive field of the photodiodeand a height equal to the depth of the channel above the photodiode.

The signals from the photodiodes are carried via output lines 76 to theprocessor (not shown in FIGS. 3A and 3B), which processes the signalsinto values corresponding to the length of the polynucleotide givingrise to the signal. The processor then uses this information to controlactive elements in the discrimination region.

With reference to FIG. 3B, the photodiode 78 can be larger in diameterthan the width of the main channel, forming a detection region 80 thatis longer (along the length of the main channel 82) than it is wide. Thevolume of such a detection region is approximately equal to thecross-sectional area of the channel above the diode multiplied by thediameter of the diode.

The detection region is connected by the main channel to thediscrimination region. The discrimination region may be locatedimmediately downstream of the detection region, or may be separated by asuitable length of channel. Constraints on the length of channel betweenthe detection and discrimination regions are discussed below, withrespect to the operation of the device. This length is typically betweenabout 1 μm and about 2 cm.

The discrimination region is at the junction of the main channel and thebranch channels. It comprises the physical location where molecules aredirected into a selected branch channel. The means by which themolecules are directed into a selected branch channel may (i) be presentin the discrimination region, as in, e.g., electrophoretic ormicrovalve-based discrimination, or (ii) be present at a distantlocation, as in, e.g., electroosmotic or flow stoppage-baseddiscrimination. The following paragraphs illustrate examples ofdiscrimination regions employing such discrimination means.

FIG. 4A shows an electrophoretic discrimination means, with adiscrimination region 102 at the junction of the main channel 104 andtwo branch channels 106 and 108. The region includes electrodes 110 and112 connected to leads 114 and 116, which are in turn connected to avoltage source (not shown) incorporated into or controlled by theprocessor (not shown). The distance between the electrodes is preferablyless than the average distance between molecules in the main channel.The dimensions of the electrodes are typically on the same order as thedimensions of the channels in which they are positioned.

Electroosmotic discrimination, as is described more fully with respectto the operation of the device, below, is preferably employed withanalysis units having only two branch channels. FIG. 4B shows adiscrimination region 122 at the junction of the main channel 124 andtwo branch channels 126 and 128. The discrimination means includes anelectrode at the sample inlet of the main channel, such as electrode130, and electrodes at the sample outlets of the branch channels, suchas electrodes 132 and 134.

In another embodiment of the invention, the polynucleotides are directedinto a selected branch channel via a valve in the discrimination region.An exemplary valve is shown in FIG. 4C. The valve consists of a thinextension of material 140 to which a charge can be applied via electrodelead 142. The extension 140 can then be deflected to close one or theother of the branch channels by application of a voltage acrosselectrodes 144 and 146.

FIG. 4D shows an embodiment employing flow stoppage in one or morebranch channels as a discrimination means. The sample solution is movedthrough such a device by application of positive pressure at thesolution inlet. Discrimination or routing of the polynucleotidemolecules is affected by simply blocking branch channels or branchchannel sample outlets into which the sample is not supposed to go, andleaving a single selected outlet open.

IV. Microfabricated Polynucleotide Analysis Device

Also included in the present invention is a microfabricatedpolynucleotide analysis device, suitable for quantitation and analysisof the size distribution of polynucleotide fragments in solution. Such adevice is a simplified version of the sorting device described above, inthat analysis units in the device do not contain a discrimination regionor branch channels, and the device does not contain a means fordirecting molecules to selected branch channels. Each analysis unitcomprises a single main channel containing a detection region asdescribed above.

Since the optics which collect the optical signal (e.g., fluorescence)can be situated immediately adjacent the flow stream (e.g., diodeembedded in the channel of a microscope objective adjacent a glasscoverslip covering the channel), the signal-to-noise ratio of the signalcollected using a microfabricated polynucleotide analysis device of theinvention is high relative to other types of devices. Specifically, thepresent methods allow, e.g., the use of oil-immersion high numericalapperature (N.A.) microscope objectives to collect the light (e.g., 1.4N.A.). Since the collection of light is proportional to the square ofthe N.A., a 1.4 N.A. objective provides about a four-fold better signalthan an 0.8 N.A. objective.

V. Microfabricated Cell Sorting Device

The present invention also includes a microfabricated device for sortingreporter-labelled cells by the level of reporter they contain. Thedevice is similar to polynucleotide-sorting device described above, butis adapted for handling particles on the size scale of cells rather thanmolecules. This difference is manifested mainly in the dimensions of themicrofabricated channels, detection and discrimination regions.Specifically, the channels in the device are typically between about 20μm and about 500 μm in width and between about 20 μm and about 500 μm indepth, to allow for an orderly flow of cells in the channels. Similarly,the volume of the detection region in a cell sorting device is largerthan that of the polynucleotide-sorting device, typically being in therange of between about 10 p1 and 100 nl.

To prevent the cells from adhering to the sides of the channels, thechannels (and coverslip) preferably contain a coating which minimizescell adhesion. Such a coating may be intrinsic to the material fromwhich the device is manufactured, or it may be applied after thestructural aspects of the channels have been microfabricated. Anexemplary coating has the surface properties of a material such as“TEFLON”.

The device may be used to sort any procaryotic (e.g., bacterial) oreukaryotic (e.g., mammalian) cells which can be labeled (e.g., viaantibodies) with optically-detectable reporter molecules (e.g.,fluorescent dyes). Exemplary mammalian cells include human blood cells,such as human peripheral blood mononuclear cells (PBMCs). The cells canbe labeled with antibodies directed against any of a variety of cellmarker antigens (e.g., HLA DR, CD3, CD4, CD8, CD11a, CD11c, CD14, CD16,CD20, CD45, CD45RA, CD62L, etc.), and the antibodies can in turn bedetected using an optically-detectable reporter (either viadirectly-conjugated reporters or via labelled secondary antibodies)according to methods known in the art.

It will be appreciated that the cell sorting device and method describedabove can be used simultaneously with multiple optically-detectablereporters having distinct optical properties. For example, thefluorescent dyes fluorescein (FITC), phycoerythrin (PE), and “CYCHROME”(Cy5-PE) can be used simultaneously due to their different excitationand emission spectra. The different dyes may be assayed, for example, atsuccessive detection and discrimination regions. Such regions may becascaded as shown in FIG. 7 to provide samples of cells having aselected amount of signal from each dye.

VI. Operation of a Microfabricated Polynucleotide Sorting Device

In operation of the device, a solution of reporter-labelledpolynucleotides is prepared as described below and introduced into thesample inlet end(s) of the analysis unit (s). The solution may beconveniently introduced into a reservoir, such as reservoir 48 of FIG.1, via a port or connector, such as connector 70 in FIG. 2, adapted forattachment to a segment of tubing, such as liquid chromatography or HPLCtubing.

It is typically advantageous to “hydrate” the device (i.e., fill thechannels of the device with the solvent, e.g., water or a buffersolution, in which the polynucleotides will be suspended) prior tointroducing the polynucleotide-containing solution. Such hydrating canbe achieved by supplying water or the buffer solution to the devicereservoir and applying hydrostatic pressure to force the fluid throughthe analysis unit(s).

Following such hydration, the polynucleotide-containing solution isintroduced into the sample inlets of the analysis unit(s) of the device.As the stream of polynucleotides derivatized with a detectable reporter(e.g., a fluorescent dye) is passed in a single file manner through thedetection region, the optical signal (e.g., fluorescence) from theoptically-detectable reporter moieties on each molecule are quantitatedby an optical detector and converted into a number used in calculatingthe approximate length of polynucleotide in the detection region.

Exemplary reporter moieties, described below in reference to samplepreparation, include fluorescent moieties which can be excited to emitlight of characteristic wavelengths by an excitation light source.Fluorescent moieties have an advantage in that each molecule can emit alarge number of photons (e.g., upward of 10⁶) in response to excitingradiation. Suitable light sources include lasers, laser diodes,high-intensity lamps, e.g., mercury lamps, and the like. In embodimentswhere a lamp is used, the channels are preferably shielded from thelight in all regions except the detection region, to avoid bleaching ofthe label. In embodiments where a laser is used, the laser can be set toscan across a set of detection regions from different analysis units.

Where laser diodes are used as a light source, the diodes may bemicrofabricated into the same chip that contains the analysis units(polynucleotide analysis chip; PAC). Alternatively, the laser diodes maybe incorporated into a second chip (laser diode chip; LDC) that isplaced adjacent the PAC such that the laser light from the diodes shineson the detection regions. The photodiodes in the LDC are preferablyplaced at a spacing that corresponds to the spacing of the detectionregions in the PAC.

Other optically-detectable reporter moieties include chemiluminescentmoieties, which can be used without an excitation light source.

The level of reporter signal is measured using an optical detector, suchas a photodiode (e.g., an avalanche photodiode), a fiber-optic lightguide leading, e.g., to a photomultiplier tube, a microscope with a highnumerical apperature (N.A.) objective and an intensified video camera,such as a SIT camera, or the like. The detector may be microfabricatedor placed into the PAC itself (e.g., a photodiode as illustrated inFIGS. 3A and 3B), or it may be a separate element, such as a microscopeobjective.

In cases where the optical detector is a separate element, it isgenerally necessary to provide a means to restrict the collection ofsignal from the detection region of a single analysis unit. It may alsobe advantageous to provide an automated means of scanning or moving thedetector relative to the PAC. For example, the PAC can be secured in amovable mount (e.g., a motorized/computer-controlled micromanipulator)and scanned under the objective. A fluorescence microscope, which hasthe advantage of a built-in excitation light source (epifluorescence),is preferably employed for detection of a fluorescent reporter.

Since current microfabrication technology enables the creation ofsub-micron structures employing the elements described herein, thedimensions of the detection region are influenced primarily by the sizeof the molecules under study. These molecules can be rather large bymolecular standards. For example, lambda DNA (˜50 kb) in solution has adiameter of approximately 0.5 μm. Accordingly, detection regions usedfor detecting polynucleotides in this size range have a cross-sectionalarea large enough to allow such a molecule to pass through without beingsubstantially slowed down relative to the flow of the solution carryingit and causing a “bottle neck”. The dimensions of a channel shouldtherefore be at least about twice, preferably at least about five timesas large per side or in diameter as the diameter of the largest moleculethat will be passing through it.

Another factor important to consider in the practice of the presentinvention is the optimal concentration of polynucleotides in the samplesolution. The concentration should be dilute enough so that a largemajority of the polynucleotide molecules pass through the detectionregion one by one, with only a small statistical chance that two or moremolecules pass through the region simultaneously. This is to insure thatfor the large majority of measurements, the level of reporter measuredin the detection region corresponds to a single molecule, rather thantwo individual molecules.

The parameters which govern this relationship are the volume of thedetection region and the concentration of molecules in the samplesolution. The probability that the detection region will contain two ormore molecules (P_(≧2)) can be expressed as

P _(≧2)=1−{1+[DNA]*V}*e ^(−[DNA]*v)

where [DNA] is the concentration of polynucleotides in units ofmolecules per μm³ and V is the volume of the detection region in unitsof μm³.

It will be appreciated that P_(≧2) can be minimized by decreasing theconcentration of polynucleotides in the sample solution. However,decreasing the concentration of polynucleotides in the sample solutionalso results in increased volume of solution processed through thedevice and can result in longer run times. Accordingly, the objectivesof minimizing the simultaneous presence of multiple molecules in thedetection chamber (to increase the accuracy of the sorting) needs to bebalanced with the objective of generating a sorted sample in areasonable time in a reasonable volume containing an acceptableconcentration of polynucleotide molecules.

The maximum tolerable P_(≧2) depends on the desired “purity” of thesorted sample. The “purity” in this case refers to the fraction ofsorted polynucleotides that are in the specified size range, and isinversely proportional to P_(≧2).

For example, in applications where high purity is not required, such asthe purification of a particular restriction fragment from an enzymaticdigest of a portion of vector DNA, a relatively high P_(≧2) (e.g.,P_(≧2)=0.2) may be acceptable. For most applications, maintaining P_(≧2)at or below about 0.1 provides satisfactory results.

In an example where P_(≧2) is equal 0.1, it is expected that in about10% of measurements, the signal from the detection region will be due tothe presence of two or more polynucleotide molecules. If the totalsignal from these molecules is in the range corresponding to the desiredsize fragment, these (smaller) molecules will be sorted into the channelor tube containing the desired size fragments.

The DNA concentration needed to achieve a particular value P_(≧2) in aparticular detection volume can be calculated from the above equation.For example, a detection region in the shape of a cube 1 μm per side hasa volume of 1 femptoliter (fl). A concentration of molecules resulting,on average, in one molecule per fl, is about 1.7 nM. Using a P_(≧2)value of about 0.1, the polynucleotide concentration in a sampleanalyzed or processed using such a 1 fl detection region volume isapproximately 0.85 nM, or roughly one DNA molecule per 2 detectionvolumes ([DNA]*V=˜0.5). If the concentration of DNA is such that [DNA]*Vis 0.1, P_(≧2) is less than 0.005; i.e., there is less than a one halfof one percent chance that the detection region will at any given timecontain two of more fragments.

The signal from the optical detector is routed, e.g., via electricaltraces and pins on the chip, to a processor, which processes the signalsinto values corresponding to the length of the polynucleotide givingrise to the signal. These values are then compared, by the processor, topre-loaded instructions containing information on which branch channelmolecules of a particular size range will be routed into. Following adelay period that allows the molecule from which the reporter signaloriginated to arrive at the discrimination region, the processor sends asignal to actuate the active elements in the discrimination region suchthat the molecule is routed into the appropriate branch channel.

The delay period is determined by the rate at which the molecules movethrough the channel (their velocity relative to the walls of thechannel) and the length of the channel between the detection region andthe discrimination region. In cases where the sample solution is movedthrough the device using hydrostatic pressure (applied, e.g., aspressure at the inlet end and/or suction at the outlet end), thevelocity is typically the flow rate of the solution. In cases where themolecules are pulled through the device using some other means, such asvia electroosmotic flow with an electric field set up between the inletend and the outlet end, the velocity as a function of molecule size canbe determined empirically by running standards, and the velocity for aspecific molecule calculated based on the size calculated for it fromthe reporter signal measurement.

A relevant consideration with respect to the velocity at which thepolynucleotide molecules move through the device is the shear force thatthey may be subject to. At the channel dimensions contemplated herein,the flow through the channels of the device is primarily laminar flowwith an approximately parabolic velocity profile. Since thecross-sectional area of the channels in the device can be on the sameorder of magnitude as the diameter of the molecules being analyzed,situations may arise where a portion of a particular molecule is verynear the wall of the channel, and is therefore in a low-velocity region,while another portion of the molecule is near the center of the channel,i.e., in a high-velocity region. This situation creates a shear force(F) on the molecule, which can be estimated using the followingexpression:

F=6πηR _(λ) V

where R_(λ)is the radius of the molecule and η is the viscosity of thesolution. This expression assumes that the molecule is immobilized on astationary surface and subject to uniform flow of velocity V.

The amount of force necessary to break a double-stranded fragment of DNAis approximately 100 pN. Accordingly, the maximal shear force that themolecules are subjected to should preferably be kept below this value.Substituting appropriate values for the variables in the aboveexpression for lambda DNA yields a maximum velocity of about 1 cm/secfor a channel 1 μm in radius (i.e., a channel of a dimension where oneportion of the lambda molecule can be at or near the wall of the channelwith the opposite side in the center of the channel). Since devicesdesigned for use with such large molecules will typically have channelsthat are considerably larger in diameter, the maximum “safe” velocitywill typically be greater than 1 cm/sec.

As discussed above, the sample solution introduced into a device of theinvention should be dilute enough such that there is a high likelihoodthat only a single molecule occupies the detection region at any giventime. It follows then that as the solution flows through the devicebetween the detection and discrimination regions, the molecules will bein “single file” separated by stretches of polynucleotide-free solution.The length of the channel between the detection and discriminationregion should therefore not be so long as to allow random thermaldiffusion to substantially alter the spacing between the molecules. Inparticular, the length should be short enough that it can be traversedin a time short enough such that even the smallest molecules beinganalyzed will typically not be able to diffuse and “switch places” inthe line of molecules.

The diffusion constant of a 1 kb molecule is approximately 5 μm²/sec;the diffusion equation gives the distance that the molecule diffuses intime t as:

<x ² >˜Dt

Using this relationship, it can be appreciated that a 1 kbp fragmenttakes about 0.2 seconds to diffuse 1 μm. The average spacing ofmolecules in the channel is a function of the cross-sectional area ofthe channel and the molecule concentration, the latter being typicallydetermined in view of acceptable values of P_(≧2) (see above). From theabove relationships, it is then straightforward to calculate the maximumchannel length between the detection and discrimination region whichwould ensure that molecules don't “switch places”. In practice, thechannel length between the detection and discrimination regions isbetween about 1 μm and about 2 cm.

As illustrated above with respect to FIGS. 4A, 4B 4C and 4D, there are anumber of ways in which molecules can be routed or sorted into aselected branch channel. For example, in a device employing thediscrimination region shown in FIG. 4A, the solution is preferably movedthrough the device by hydrostatic pressure. Absent any field appliedacross electrodes 110 and 112, a molecule would have an equalprobability of entering one or the other of the two branch channels 106and 108. The sorting is accomplished by the processor temporarilyactivating a voltage source connected to the electrode leads 114 and 116just before or at the time the molecule to be routed enters the junctionof the main channel and the two branch channels. The resulting electricfield exerts a force on the negatively-charged DNA molecule, biasing ittoward the positively-charged electrode. The molecule will then becarried down the branch channel containing the positively-chargedelectrode by the bulk solution flow. The electric field is turned offwhen the molecule has committed itself to the selected channel. As soonas the molecule clears the corner from the discrimination region andinto the branch channel, it escapes effects of the electric field thatwill be applied to the next molecule in the solution stream.

The discrimination region shown in FIG. 4B is designed for use in adevice that employs electroosmotic flow, rather than flow induced byhydrostatic pressure, to move both the polynucleotides and bulk solutionthrough the device. Electrodes are set up in the channels at the inletand outlet ends of the device. Application of an electric field at theends of the channels (with electrode 130 being negative, and electrodes132 and 134 being positive) sets up bulk solution flow according towell-established principles of electroosmotic flow (see, e.g., Baker,1995). When a specific polynucleotide molecule enters the junctionregion between the main channel and the two branch channels, the voltageto one of either electrodes 132 or 134 is shut off, leaving a singleattractive force, acting on the solution and the DNA molecule, into theselected branch channel. As above, both branch channel electrodes areactivated after the molecule has committed to the selected branchchannel in order to continue bulk flow through both channels.

In another embodiment of the invention, shown in FIG. 4C, thepolynucleotides are directed into a selected branch channel via a valvein the discrimination region. An exemplary valve is shown in FIG. 4C.The valve consists of a thin extension of material 140 which can becharged via an electrode 142. The extension can then be deflected toclose one or the other of the branch channels by application of anappropriate voltage across electrodes 144 and 146. As above, once themolecule has committed, the voltage can be turned off.

In a device in which the sample solution is moved through the device byapplication of positive pressure at the sample inlet end(s) of theanalysis unit(s), the discrimination function may be affected by simplyblocking branch channel sample outlets into which the sample is notsupposed to go, and leaving the selected outlet open. Due to the smallsize scale of the channels and the incompressibility of liquids,blocking the solution flow creates an effective “plug” in the unselectedbranch channels, routing the molecule along with the bulk solution flowinto the selected channel. This embodiment is illustrated in FIG. 4D. Itcan be achieved by, for example, incorporating valve structuresdownstream of the discrimination region.

Alternatively, the discrimination function may be affected by changingthe hydrostatic pressure at the sample outlets of the branch channelsinto which the sample is not supposed to go. Specifically, if the branchchannels in a particular analysis unit all offer the same resistance tofluid flow, and the pressure at the sample inlet of the main channel ofan analysis unit is P, then the fluid flow out of any selected branchchannel can be stopped by applying a pressure P/n at the sample outletof that branch channel, where n is the number of branch channels in thatanalysis unit. Accordingly, in an analysis unit having 2 branchchannels, the pressure applied at the outlet of the branch to be blockedis P/2.

It will be appreciated that the position and fate of the molecules inthe discrimination region can be monitored by additional detectionregions installed, e.g., immediately upstream of the discriminationregion and/or in the branch channels immediately downstream of thebranch point. This information be used by the processor to continuouslyrevise estimates of the velocity of the molecules in the channels and toconfirm that molecules having selected size characteristics end up inthe selected branch channel.

Solution from the branch channels is collected at the outlet ends of theanalysis units. As described above, devices with a plurality of analysisunits typically collect the solution from corresponding branch channelsof each unit into a manifold, which routes the solution flow to anoutlet port, which can be adapted for receiving, e.g., a segment oftubing or a sample tube, such as a standard 1.5 ml centrifuge tube.

The time required to isolate a desired quantity of polynucleotidedepends on a number of factors, including the size of thepolynucleotide, the rate at which each analysis unit can process theindividual fragments, and the number of analysis units per chip, and canbe easily calculated using basic formulas. For example, a chipcontaining 1000 analysis units, each of which can sort 1000 fragmentsper second, could isolate 0.1 μg of 10 kb DNA in about 2.5 hours.

VII. Operation of Other Microfabricated Devices of the Invention

Operation of a microfabricated cell sorting device is essentially asdescribed above with respect to the polynucleotide sorting device. Sincecells typically do not have predictable a net charge, the directingmeans are preferably ones employing a valve in the discrimination regionas described above, or flow stoppage, either by valve or hydrostaticpressure.

Operation of a microfabricated analysis device is accomplishedessentially as is described above, except that functions relating tosorting polynucleotide molecules into branch channels don't need to beperformed. The processor of such analysis devices is typically connectedto a data storage unit, such as computer memory, hard disk or the like,as well as to a data output unit, such as a display monitor, printerand/or plotter. The sizes of the polynucleotide molecules passingthrough the detection region are calculated and stored in the datastorage unit. This information can then be further processed and/orrouted to the data output unit for presentation as, e.g., histograms ofthe size distribution of DNA molecules in the sample. The data can, ofcourse, be presented in real time as the sample is flowing through thedevice, allowing the practitioner of the invention to continue the runonly as long as is necessary to obtain the desired information.

VIII. Microfabrication of Devices

Analytical devices having microscale flow channels, valves and otherelements can be designed and fabricated from a solid substrate material.Silicon is a preferred substrate material because of the welldevelopedtechnology permitting its precise and efficient fabrication, but othermaterials may be used, including polymers such aspolytetrafluoroethylenes. Micro-machining methods well known in the artinclude film deposition processes, such as spin coating and chemicalvapor deposition, laser fabrication or photolithographic techniques, oretching methods, which may be performed by either wet chemical or plasmaprocesses. (See, for example, Angell et al., Scientific American248:44-55 (1983) and Manz et al., Trends in Analytical Chemistry 10:144-149 (1991), all incorporated herein by reference).

FIGS. 5A-5D illustrate the initial steps in microfabricating thediscrimination region portion of a nucleic acid sorting device 20(FIG. 1) by photolithographic techniques. As shown, the structureincludes a silicon substrate 160. The silicon wafer which forms thesubstrate is typically washed in a 4:1 H₂SO₄/H₂O₂ bath, rinsed in waterand spun dry. A layer 162 of silicon dioxide, preferably about 0.5 μm inthickness, is formed on the silicon, typically by heating the siliconwafer to 800-1200° C. in an atmosphere of steam. The oxide layer is thencoated with a photoresist layer 164, preferably about 1 μm in thickness.Suitable negative-or positive-resist materials are well known. Commonnegative-resist materials include two-component bisarylazide/rubberresists, and positive-resist materials include polymethylmethacrylate(PMMA) and two-component diazoquinone/phenolic resin materials. See,e.g., “Introduction to Microlithography”, Thompson, L. F. et al., eds.,ACS Symposium Series, Washington D.C. (1983).

The coated laminate is irradiated through a photomask 166 imprinted witha pattern corresponding in size and layout to the desired pattern of themicrochannels. Methods for forming photomasks having desired photomaskpatterns are well known. For example, the mask can be prepared byprinting the desired layout on an overhead transparency using a highresolution (3000 dpi) printer. Exposure is carried out on standardequipment such as a Karl Suss contact lithography machine.

In the method illustrated in FIGS. 5A-5D, the photo-resist is a negativeresist, meaning that exposure of the resist to a selected wavelength,e.g., UV, light produces a chemical change that renders the exposedresist material resistant to the subsequent etching step. Treatment witha suitable etchant removes the unexposed areas of the resist, leaving apattern of bare and resist-coated silicon oxide on the wafer surface,corresponding to the layout and dimensions of the desiredmicrostructures. In the present example, because a negative resist wasused, the bare areas correspond to the printed layout on the photomask.

The wafer is now treated with a second etchant material, such as areactive ion etch (RIE), effective to dissolve the exposed areas ofsilicon dioxide. The remaining resist is removed, typically with hotaqueous H₂SO₄. The remaining pattern of silicon dioxide (162) now servesas a mask for the silicon (160). The channels are etched in the unmaskedareas of the silicon substrate by treating with a KOH etching solution.Depth of etching is controlled by time of treatment. Additionalmicro-components may also be formed within the channels by furtherphotolithography and etching steps, as discussed below.

Depending on the method to be used for directing the flow of moleculesthrough the device, electrodes and/or valves are fabricated into theflow channels. A number of different techniques are available forapplying thin metal coatings to a substrate in a desired pattern. Theseare reviewed in, for example, Krutenat, Kirk-Othmer 3rd ed., Vol. 15,pp. 241-274, incorporated herein by reference. A convenient and commontechnique used in fabrication of microelectronic circuitry is vacuumdeposition. For example, metal electrodes or contacts may be evaporatedonto a substrate using vacuum deposition and a contact mask made from,e.g., a “MYLAR” sheet. Various metals such as platinum, gold, silver orindium/tin oxide (ITO) may be used for the electrodes.

Deposition techniques allowing precise control of the area of depositionare preferred for application of electrodes to the side walls of thechannels in the device. Such techniques are described, for example, inKrutenat, above, and references cited therein. They include plasmaspraying, where a plasma gun accelerates molten metal particles in acarrier gas towards the substrate, and physical vapor deposition usingan electron beam, where atoms are delivered on line-of-sight to thesubstrate from a virtual point source. In laser coating, a laser isfocused onto the target point on the substrate, and a carrier gasprojects powdered coating material into the beam, so that the moltenparticles are accelerated toward the substrate.

Another technique allowing precise targeting uses an electron beam toinduce selective decomposition of a previously deposited substance, suchas a metal salt, to a metal. This technique has been used to producesub-micron circuit paths (e.g., Ballantyne et al., 1973).

When pressure separation is to be used for discrimination of molecules,valves are used to block or unblock the pressurized flow of moleculesthrough selected channels. A thin cantilever, for example, may beincluded within a branch point, as shown in FIG. 6B, such that it may bedisplaced towards one or the other wall of the main channel, typicallyby electrostatic attraction, thus closing off a selected branch channel.Electrodes are provided, as described above, on the walls of the channeladjacent to the end of the cantilever, as are suitable electricalcontacts for applying a potential to the cantilever. Because thecantilever in FIG. 6B is parallel to the direction of etching, it may beformed of a thin layer of silicon by incorporating the element into theoriginal photoresist pattern. The cantilever is preferably coated with adielectric material such as silicon nitride, as described in Wise, etal., 1995, for example, to prevent short circuiting between theconductive surfaces.

In a separate embodiment, illustrated in FIG. 4D, a valve is situatedwithin each branch channel, rather than at the branch point, to closeoff and terminate pressurized flow through selected channels. Becausethe valves are located downstream of the discrimination region, thechannels in this region may be formed having a greater width than in thediscrimination region, which simplifies the formation of valves.

Such a valve within a channel may be microfabricated, if necessary, inthe form of an electro-statically operated cantilever or diaphragm.Techniques for forming such elements are well known; see, for example,Wise, et al., 1995; Aine, et al., 1986; Gravesen, et al., 1995;O'Connor, 1986; and van Lintel, 1993, incorporated herein by reference.Typical processes include the use of selectively etched sacrificiallayers in a multilayer structure, or the undercutting of a layer of,e.g., silicon dioxide, via anisotropic etching. For example, to form acantilever within a channel, as illustrated in FIGS. 6A-6B, asacrificial layer 168 may be formed adjacent to a small section of anon-etchable material 170, using known photolithography methods, on thebottom floor of a channel, as shown in FIG. 6A. Both layers are thencoated with, for example, silicon dioxide or another non-etchable layer,as shown at 172. Etching of the sacrificial layer leaves thecantilevered member 174 within the channel, as shown in FIG. 6B.Suitable materials for the sacrificial layer, non-etchable layers andetchant, respectively, include undoped silicon, p-doped silicon andsilicon dioxide, and the etchant EDP (ethylene diamine/pyrocatechol).

The width of the cantilever or diaphragm should approximately equal thatof the channel, allowing for movement within the channel. If desired,the element may be coated with a more malleable material, such as ametal, to allow for a better seal. Such coating may also be employed torender a non-conductive material, such as silicon dioxide, conductive.

As above, suitable electrical contacts are provided for displacing thecantilever or diaphragm towards the opposing surface of the channel.When the upper surface is a glass cover plate, as described below,electrodes and contacts may be deposited onto the glass.

It will be apparent to one of skill in the field that other types ofvalves or switches could be designed and fabricated, using well knownphotolithographic or other microfabrication techniques, for controllingflow within the channels of the device. Multiple layers of channelscould also be prepared.

Operation of the valves or charging of the electrodes, in response tothe level of fluorescence measured from an analyte molecule, iscontrolled by the processor, which receives this information from thedetector. All of these components are operably connected in theapparatus, and electrical contacts are included as necessary, usingstandard microchip circuitry.

In preferred embodiments, an integrated semiconductor laser and/or anintegrated photodiode detector are included on the silicon wafer in thevicinity of the detection region. This design provides the advantages ofcompactness and a shorter optical path for exciting and/or emittedradiation, thus minimizing distortion.

The silicon substrate containing the microfabricated flow channels andother components is covered and sealed, preferably with a thin glass orquartz cover, although other clear or opaque cover materials may beused. When external radiation sources or detectors are employed, theinterrogation region is covered with a clear cover material to allowoptical access to the analyte molecules. Anodic bonding to a “PYREX”cover slip may be accomplished by washing both components in an aqueousH₂SO₄/H₂O₂ bath, rinsing in water, and then heating to about 350° C.while applying a voltage of, e.g., 450V.

IV. Polynucleotide Sample Preparation

Polynucleotide samples are prepared by labeling the polynucleotide to beanalyzed or sorted with a suitable optically-detectable reporter. Thereporter associates with or incorporates into the DNA such that thereporter signal from a particular DNA molecule is proportional to thelength of that DNA molecule.

Fluorescent dyes, particularly ones that intercalate into thepolynucleotide backbone, comprise an exemplary set ofoptically-detectable reporters. There are a number of known dyes whichselectively bind to polynucleotides. These include, but are not limitedto, Hoechst 33258, Hoechst 33342, DAPI (4′,6-diamidino-2-phenylindoleHCl), propidium iodide, dihydroethidium, acridine orange, ethidiumbromide, ethidium homodimers (e.g., EthD-1, EthD-2), acridine-ethidiumheterodimer (AEthD) and the thiazole orange derivatives PO-PRO, BO-PRO,YO-PRO, TO-PRO, as well as their dimeric analogs POPO, BOBO, YOYO, andTOTO. The dimeric analogs, especially YOYO-1 and TOTO-1, are particularsuitable for use with the present invention due to their high bindingaffinity for nucleic acids, which results in extremely high detectionsensitivity. All of these compounds can be obtained from MolecularProbes (Eugene, Oreg.). Extensive information on their spectralproperties, use, and the like is provided in Haugland, 1992,incorporated herein by reference.

The dyes bind at a maximum density of about one dye molecule per fivebase pairs. Thus, by measuring the fluorescence intensity of a molecule,its length can be determined, with a resolution of five base pairs. Fora 5 Kbp molecule, this corresponds to a resolution of about 0.1%, ascompared to about 10% typically obtained with gel electrophoresis.

The polynucleotide mixture is diluted in distilled water or a suitablebuffer, such as TE buffer, to a concentration determined using theconsiderations described above. Dye is added in a 5:1 base pair/dyemolecule stoichiometry.

IV. Additional Embodiments

FIG. 7 shows a device with analysis units containing a cascade ofdetection and discrimination regions suitable for successive rounds ofpolynucleotide or cell sorting. Such a configuration may be used, forexample, with a polynucleotide sorting device to generate a series ofsamples containing “fractions” of polynucleotides, where each fractioncontains a specific size range of polynucleotide fragments (e.g., thefirst fraction contains 100-500 bp fragments, the next 500-1000 bpfragments, and so on). In a cell sorting device, such a cascadeconfiguration may be used to sequentially assay the cell for, e.g., 3different fluorescent dyes corresponding to expression of threedifferent molecular markers. Samples collected at the outlets of thedifferent branch channels contain pools of cells expressing definedlevels of each of the three markers.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

It is claimed:
 1. A device for processing a flow of polynucleotidemolecules comprising a chip having a substrate and an analysis unit,wherein the analysis unit comprises: a) a main channel having apolynucleotide sample inlet, a detection region downstream of the sampleinlet, and a branch point discrimination region adjacent to anddownstream of the detection region, wherein on average onepolynucleotide molecule at a time is placed within the detection region;(b) at least two branch channels originating at the branch pointdiscrimination region and in communication with the main channel; (c) adetector sensitive to each polynucleotide molecule passing through thedetection region; and (d) a flow control responsive to the detector andacting to direct each polynucleotide molecules at the discriminationregion into a selected branch channel.
 2. A device of claim 1, whereinthe channels are between about 1 and 10 micrometers in width and betweenabout 1 and 10 micrometers in depth.
 3. A device of claim 1, wherein thedetection region has a volume of between about 1 femtoliter and 1picoliter.
 4. A device of claim 1, further comprising a transparentcover slip bonded to the substrate and covering the channels.
 5. Adevice of claim 1, wherein the detector comprises a microscope thatmeasures the level of a reporter associated with the polynucleotidemolecules.
 6. A device of claim 5, wherein the microscope is afluorescence microscope and excites a fluorescent reporter.
 7. A deviceof claim 5, wherein the microscope comprises a high numerical apertureobjective.
 8. A device of claim 7, wherein the microscope objective hasa numerical aperture of about 1.4.
 9. A device of claim 1, furthercomprising a laser that excites a reporter associated with thepolynucleotide molecules and the detector comprises an integratedphotodiode that measures the level of reporter.
 10. A device of claim 1,wherein the polynucleotide molecules are associated with an opticallydetectable reporter.
 11. A device of claim 10, wherein the reporter isat least one of fluorescent and chemiluminescent.
 12. A device of claim10, wherein the reporter is at least one fluorescent molecule selectedfrom the group consisting of POPO, BOBO, YOYO, and TOTO.
 13. A device ofclaim 1, wherein each polynucleotide molecule is directed to a selectedbranch channel based on a measured level of reporter corresponding tothe size of the polynucleotide molecule.
 14. A device of claim 1,wherein the flow control comprises a pair of electrodes that apply anelectric field across the discrimination region and into a branchchannel.
 15. A device of claim 1, wherein the flow control comprises apressure differential, and wherein the flow of polynucleotide moleculesis directed by adjusting the pressure at the outlet of each branchchannel to allow or curtail flow through the channel.
 16. A device ofclaim 1, wherein the detector is an optical detector having a shortoptical path length.
 17. A device of claim 1, further comprising asample reservoir.
 18. A device of claim 1, further comprising amicroprocessor in communication with at least one of the detector andthe flow control.
 19. A device of claim 1, wherein the flow ofpolynucleotide molecules is directed by at least onre pump and at leastone valve.
 20. A device of claim 1, wherein each dimension of eachchannel is at least twice as large as the diameter of the largestpolynucleotide molecule to be processed.
 21. A device of claim 1,wherein each dimension of each channel is at least five times as largeas the diameter of the largest polynucleotide molecule to be processed.22. A device of claim 1, wherein the probability that the detectionregion contains two or more polynucleotide molecules is at or belowabout 0.1.
 23. A device of claim 1, wherein the detection region has adetection volume defined by channel dimensions and the detector, andwherein the concentration of polynucleotide molecules in the flow isfrom about 1 molecule per two detection volumes to about 1 molecule perten detection volumes.
 24. A device of claim 1, wherein the channellength between the detection region and the discrimination region isbetween about 1 micrometer and 2 centimeters.
 25. A device of claim 1,Wherein the substrate comprises at least one of silicon and a polymer.26. A device of claim 1, wherein the analysis unit is microfabricatedonto the substrate.
 27. A device of claim 26, wherein the analysis unitfurther comprises a flow that places, on average, one polynucleotidemolecule at a time within the detection region.
 28. A device of claim 1,wherein the analysis unit further comprises a flow that places, onaverage, one polynucleotide molecule at a time within the detectionregion.
 29. A device for processing a flow of polynucleotide moleculescomprising a chip having a substrate and an analysis unit, wherein theanalysis unit comprises: (a) a main channel having a polynucleotidesample inlet, a detection region downstream of the sample inlet, and abranch point discrimination region adjacent to and downstream of thedetection region, wherein on average one polynucleotide molecule at atime is placed within the detection region; (b) at least two branchchannels originating at the branch point discrimination region and incommunication with the main channel; (c) a detector sensitive to eachpolynucleotide molecule passing through the detection region; and (d) aflow control responsive to the detector and comprising a pair ofelectrodes that apply an electric field across the discrimination regionand into a branch channel.
 30. A device for processing a flow ofpolynucleotide molecules comprising a chip having a substrate and ananalysis unit, wherein the analysis unit comprises: (a) a main channelhaving a polynucleotide sample inlet, a detection region downstream ofthe sample inlet, and a branch point discrimination region adjacent toand downstream of the detection region, wherein on average onepolynucleotide molecule at a time is placed within the detection region;(b) at least two branch channels originating at the branch pointdiscrimination region and in communication with the main channel; (c) adetector sensitive to each polynucleotide molecule passing through thedetection region; and (d) a flow control responsive to the detector andcomprising a pressure differential, wherein the flow of polynucleotidemolecules is directed by adjusting the pressure at the outlet of eachbranch channel to allow or curtail flow through the channel.
 31. Adevice for processing a flow of polynucleotide molecules comprising achip having a substrate and an analysis unit, wherein the analysis unitcomprises: (a) a main channel having a polynucleotide sample inlet, adetection region downstream of the sample inlet, and an outlet regionadjacent to and downstream of the detection region; (b) a detectorsensitive to each polynucleotide passing through the detection region,wherein on average one polynucleotide molecule at a time is placedwithin the detection region.
 32. A device of claim 31, wherein thedetector is sensitive to the size of each polynucleotide moleculepassing through the detection region.
 33. A device of claim 31, whereinthe detector is sensitive to a reporter associated with eachpolynucleotide molecule.
 34. A device of claim 31, wherein the detectorcomprises an optical detector.
 35. A device of claim 34, wherein theoptical detector comprises a microscope.
 36. A device of claim 35,wherein the microscope comprises a high numerical aperture objective.37. A device of claim 36, wherein the microscope objective has anumerical aperture of about 1.4.
 38. A device of claim 31, wherein eachdimension of each channel is at least twice as large as the diameter ofthe largest polynucleotide molecule to be processed.
 39. A device ofclaim 31, wherein each dimension of each channel is at least five timesas large as the diameter of the largest polynucleotide molecule to beprocessed.
 40. A device of claim 31, wherein the probability that thedetection region contains two or more polynucleotide molecules is at orbelow about 0.1.
 41. A device of claim 31, wherein the detection regionhas a detection volume defined by channel dimensions and the detector,and wherein the concentration of polynucleotide molecules in the flow isfrom about 1 molecule per two detection volumes to about 1 molecule perten detection volumes.
 42. A device of claim 31, wherein the substratecomprises at least one of silicon and a polymer.
 43. A device of claim31, wherein the flow of polynucleotide molecules is directed by at leastone pump and at least one valve.
 44. A device of claim 31, wherein theanalysis unit is microfabricated onto the substrate.
 45. A device ofclaim 44, wherein the analysis unit further comprises a flow thatplaces, on average, one polynucleotide molecule at a time within thedetection region.
 46. A device of claim 31, wherein the analysis unitfurther comprises a flow that places, on average, one polynucleotidemolecule at a time within the detection region.